The present invention relates generally to improved methods of making recombinant proteins using one or more apoptosis inhibitors.
Control of cell numbers in mammals is believed to be determined, in part, by a balance between cell proliferation and cell death. One form of cell death, sometimes referred to as necrotic cell death, is typically characterized as a pathologic form of cell death resulting from some trauma or cellular injury. In contrast, there is another, xe2x80x9cphysiologicxe2x80x9d form of cell death which usually proceeds in an orderly or controlled manner. This orderly or controlled form of cell death is often referred to as xe2x80x9capoptosisxe2x80x9d [see, e.g., Barr et al., Bio/Technology, 12:487-493 (1994); Steller et al., Science, 267:1445-1449 (1995)]. Apoptotic cell death naturally occurs in many physiological processes, including embryonic development and clonal selection in the immune system [Itoh et al., Cell, 66:233-243 (1991)].
Control of cell numbers in cell culture and bioreactors is also a balance between cell proliferation and cell death. There have been reports in the literature indicating cell death in bioreactors can be an apoptotic process [Suzuki E., et al., Cytotechnology, 23:55-59 (1997); Al-Rubeai, M. and Singh R. P, Curr. Opin. Biotech, 9:152-156 (1998)]. It has been described that the apoptotic process may be induced by nutrient deprivation [Franek F. and Chlxc3xa1dkova-{haeck over (S)}rxc3xa1mkovxc3xa1 K., Cytotechnology, 18:113-117 (1995); Mercille S. and Massie B., Biotechnol. Bioeng., 44:1140-1154 (1994); Singh R. P., et al., Biotechnol. Bioeng., 44:720-726 (1994)], serum deprivation [Singh R. P., et al., Biotechnol. Bioeng., 44:720-726 (1994); Zanghi A., et al., Biotech. Bioeng., 64:108-119 (1999)] or other controllable parameters of cell culture in bioreactors, but is not controlled fully because of bioreactor mechanics, a lack of full understanding of necessary culture parameters, or other undetermined causes.
As presently understood, the apoptosis or cell death program contains at least three important elementsxe2x80x94activators, inhibitors, and effectors; in C. elegans, these elements are encoded respectively by three genes, Ced-4 4, Ced-9 and Ced-3 [Steller, Science, 267:1445 (1995); Chinnaiyan et al., Science, 275:1122-1126 (1997); Wang et al., Cell, 90:1-20 (1997)]. Two of the TNFR family members, TNFR1 and Fas/Apo1 (CD95), can activate apoptotic cell death [Chinnaiyan and Dixit, Current Biology, 6:555-62 (1996); Fraser and Evan, Cell; 85:781-784 (1996)]. TNFR1 is also known to mediate activation of the transcription factor, NF-KB [Tartaglia et al., Cell, 74:845-853 (1993); Hsu et al., Cell, 84:299-308 (1996)]. In addition to some ECD homology, these two receptors share homology in their intracellular domain (ICD) in an oligomerization interface known as the death domain [Tartaglia et al., supra; Nagata, Cell, 88:355 (1997)]. Death domains are also found in several metazoan proteins that regulate apoptosis, namely, the Drosophila protein, Reaper, and the mammalian proteins referred to as FADD/MORT1, TRADD, and RIP [Cleaveland and Ihle, Cell, 81:479-482 (1995)].
Upon ligand binding and receptor clustering, TNFR1 and CD95 are believed to recruit FADD into a death-inducing signaling complex. CD95 purportedly binds FADD directly, while TNFR1 binds FADD indirectly via TRADD [Chinnaiyan et al., Cell, 81:505-512 (1995); Boldin et al., J. Biol. Chem., 270:387-391 (1995); Hsu et al., supra; Chinnaiyan et al., J. Biol. Chem., 271:4961-4965 (1996)]. It has been reported that FADD serves as an adaptor protein which recruits the Ced-3-related protease, MACH-alpha/FLICE (caspase 8), into the death signaling complex [Boldin et al., Cell, 85:803-815 (1996); Muzio et al., Cell, 85:817-827 (1996)]. MACH-alpha/FLICE appears to be the trigger that sets off a cascade of apoptotic proteases, including the interleukin-1beta converting enzyme (ICE) and CPP32/Yama, which may execute some critical aspects of the cell death programme [Fraser and Evan, supra].
It was recently disclosed that programmed cell death involves the activity of members of a family of cysteine proteases related to the C. elegans cell death gene, ced-3, and to the mammalian IL-1-converting enzyme, ICE. The activity of the ICE and CPP32/Yama proteases can be inhibited by the product of the cowpox virus gene, crmA [Ray et al., Cell, 69:597-604 (1992); Tewari et al., Cell, 81:801-809 (1995)]. Recent studies show that CrmA can inhibit TNFR1- and CD95-induced cell death [Enari et al., Nature, 375:78-81 (1995); Tewari et al., J. Biol. Chem., 270:3255-3260 (1995)].
As reviewed recently by Tewari et al., TNFR1, TNFR2 and CD40 modulate the expression of proinflammatory and costimulatory cytokines, cytokine receptors, and cell adhesion molecules through activation of the transcription factor, NF-KB [Tewari et al., Curr. Op. Genet. Develop., 6:39-44 (1996)]. NF-KB is the prototype of a family of dimeric transcription factors whose subunits contain conserved Rel regions [Verma et al., Genes Develop., 9:2723-2735 (1996); Baldwin, Ann. Rev. Immunol., 14:649-681 (1996)]. In its latent form, NF-KB is complexed with members of the IKB inhibitor family; upon inactivation of the IKB in response to certain stimuli, released NF-KB translocates to the nucleus where it binds to specific DNA sequences and activates gene transcription.
For recent reviews of such signaling pathways, see, e.g., Ashkenazi et al., Science, 281:1305-1308 (1998); Nagata, Cell, 88:355-365 (1997).
To date, there have been conflicting reports as to the effects of caspase inhibitors and expression of anti-apoptotic genes on cultured recombinant cells. For instance, Murray et al., Biotech. Bioeng., 51:298-304 (1996) describe that overexpression of bcl-2 in NSO myeloma cells failed to affect the decline phase characteristics of the cultured cells. Other investigators have found, in contrast, that bcl-2 can be effective in preventing different cell lines from death under cell-culture conditions [see, e.g., Itoh et al., Biotechnol. Bioeng., 48:118-122 (1995); Mastrangelo et al., TIBTECH, 16:88-95 (1998); Simpson et al., Biotechnol. Bioeng., 54:1-16 (1997); Singh et al., Biotechnol. Bioeng., 52:166-175 (1996)]. Goswami et al., Biotechnol. Bioeng., 62:632-640 (1999) report that they found that the caspase inhibitor, z-VAD-fmk, was unable to substantially extend the life of a serum-free culture of CHO cells.
The present invention is based on Applicants"" findings that employing one or more apoptosis inhibitor(s) in recombinant cell culturing and protein production can markedly reduce apoptosis in the cell culture and improve recombinant protein production techniques. The methods disclosed in present application are useful, for example, in prolonging cell viability in cell cultures or improving or enhancing yield of the recombinant proteins from the cell cultures. Further improvements provided by the invention are described in detail below.
In one embodiment, the invention provides a method of making recombinant proteins using one or more apoptosis inhibitors. The method includes the steps of (a) providing a vector comprising a gene encoding an apoptosis inhibitor, (b) providing a vector comprising a gene encoding a protein of interest, (c) providing a host cell, (d) transforming or transfecting the host cell with the vectors referred to in steps (a) and (b), (e) providing cell culture media, (f) culturing the transformed or transfected host cell(s) in the culture media under conditions sufficient to express the protein of interest and the apoptosis inhibitor, and (g) recovering or purifying the protein of interest from the host cells and/or the cell culture media. Optionally, the method further includes the step of admixing an additional apoptosis inhibitor into the culture media. In the method, the respective genes encoding the apoptosis inhibitor and the protein of interest may be inserted into a single vector (e.g., co-transfected in a single vector), or alternatively, be inserted into two separate vectors. Preferably, the respective genes encoding the apoptosis inhibitor and the protein of interest are inserted into two separate vectors, each vector having a different type of selection marker from the other vector. Optionally, the method provides for transient expression of the protein of interest and stable or transient expression of the apoptosis inhibitor. Optionally, the gene encoding the apoptosis inhibitor comprises a gene encoding the caspase-9-DN protein or baculovirus p35.
In another embodiment, the method includes the steps of (a) providing a vector comprising a gene encoding a protein of interest, (b) providing a host cell comprising DNA encoding an apoptosis inhibitor, (c) transforming or transfecting the host cell(s) with the vector referred to in step (a), (d) providing cell culture media, (e) culturing the transformed or transfected host cell(s) in the culture media under conditions sufficient to express the protein of interest and the apoptosis inhibitor, and (f) recovering or purifying the protein of interest from the host cells and/or cell culture media. Optionally, the gene encoding the apoptosis inhibitor may be stably integrated into the genome of the host cell. Optionally, the method includes the further step of admixing an additional apoptosis inhibitor molecule into the culture media. Optionally, the method provides for transient expression of the protein of interest and stable or transient expression of the apoptosis inhibitor.
In another embodiment, the method includes the steps of (a) providing a vector comprising a gene encoding a protein of interest, (b) providing a host cell, (c) transforming or transfecting the host cell with the vector referred to in step (a), (d) providing cell culture media, (e) providing an apoptosis inhibitor, (f) admixing the apoptosis inhibitor into the culture media, (g) culturing the host cell(s) in the culture media under conditions sufficient to express the protein of interest, and (h) recovering or purifying the protein of interest from the host cells and/or the cell culture media. Optionally, the method provides for transient expression of the protein of interest.
In another embodiment, the method includes the steps of (a) providing a vector comprising a gene encoding an apoptosis inhibitor, (b) providing a vector comprising a gene encoding a protein of interest, (c) providing a host cell, (d) transforming or transfecting the host cell with the vectors referred to in steps (a) and (b), (e) providing cell culture media, (f) culturing the transformed or transfected host cell(s) in the culture media under conditions sufficient to express the protein of interest and the apoptosis inhibitor, and (g) freezing and subsequently thawing the host cells and/or the cell culture media. Optionally, the method further includes the step of admixing an additional apoptosis inhibitor into the culture media in steps (e) or (f). In the method, the respective genes encoding the apoptosis inhibitor and the protein of interest may be inserted into a single vector, or alternatively, be inserted into two separate vectors. Preferably, the respective genes encoding the apoptosis inhibitor and the protein of interest are inserted into two separate vectors, each vector having a different type of selection marker from the other vector. Optionally, the method provides for transient expression of the protein of interest and stable or transient expression of the apoptosis inhibitor.
In another embodiment, the method includes the steps of (a) providing a vector comprising a gene encoding a protein of interest, (b) providing a host cell comprising DNA encoding an apoptosis inhibitor, (c) transforming or transfecting the host cell(s) with the vector referred to in step (a), (d) providing cell culture media, (e) culturing the transformed or transfected host cell(s) in the culture media under conditions sufficient to express the protein of interest and the apoptosis inhibitor, and (f) freezing and subsequently thawing the host cells and/or cell culture media. Optionally, the gene encoding the apoptosis inhibitor may be stably integrated into the genome of the host cell. Optionally, the method includes the further step of admixing an additional apoptosis inhibitor molecule into the culture media. Optionally, the method provides for transient expression of the protein of interest and stable or transient expression of the apoptosis inhibitor.
In another embodiment, the method includes the steps of (a) providing a vector comprising a gene encoding a protein of interest, (b) providing a host cell, (c) transforming or transfecting the host cell with the vector referred to in step (a), (d) providing cell culture media, (e) providing an apoptosis inhibitor, (f) admixing the apoptosis inhibitor into the culture media, (g) culturing the host cell(s) in the culture media under conditions sufficient to express the protein of interest, and (h) freezing and subsequently thawing the host cells and/or the cell culture media. Optionally, the method provides for transient expression of the protein of interest.
In a still further embodiment, the invention provides for improved transfection methods wherein use of one or more apoptosis inhibitor(s) and increased concentrations of transfection reagent can be employed to increase transfection efficiency.
In an even further embodiment, the invention provides a protein of interest produced in accordance with any of the methods described herein. The protein of interest may comprise a mammalian protein or non-mammalian protein, and may optionally comprise a receptor or a ligand. In one embodiment of the invention, the protein of interest will comprise a protein which itself is capable of inducing apoptosis in mammalian or non-mammalian cells in vitro or in vivo, such as Apo-2 ligand/TRAIL, Fas ligand, or TNF-alpha.